Non-invasive methods of using spectral information in determining analyte concentrations

ABSTRACT

A non-invasive method of determining the concentration of an analyte uses Raman or fluorescence spectral information. A high-intensity band of light is applied to one side of skin tissue. The high-intensity light enters the skin tissue and generates a Raman or fluorescence signal. A Raman-generating material or fluorescence-generating material is placed in a location nearest the other side of skin tissue. The Raman-generating or fluorescence-generating material is located generally opposite of the entry of the applied high-intensity light. The Raman or fluorescence signal is collected and the analyte concentration is determined using the collected Raman signal.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 60/839,299 filed on Aug. 22, 2006 which is incorporated by referencein its entirety.

FIELD OF THE INVENTION

The present invention generally relates to a method of determining theconcentration of an analyte. More specifically, the present invention isdirected to a non-invasive method of determining the concentration of ananalyte using spectral information (e.g., Raman or fluorescence).

BACKGROUND OF THE INVENTION

The quantitative determination of analytes in body fluids is of greatimportance in the diagnoses and maintenance of certain physiologicalabnormalities. For example, lactate, cholesterol and bilirubin should bemonitored in certain individuals. In particular, determining glucose inbody fluids is important to diabetic individuals who must frequentlycheck the glucose level in their body fluids to regulate the glucoseintake in their diets. The results of such tests can be used todetermine what, if any, insulin or other medication needs to beadministered.

Determining the analyte concentration of, for example, glucose istypically performed by invasive methods. It would be desirable todetermine analyte concentrations by using a non-invasive method.

Non-invasive methods may incorporate the use of different types ofsignals to determine the analyte concentration. One type of signal is aRaman spectral signal. The use of Raman spectral information, however,has had limited application in determining non-invasive analyteconcentrations because the signals tend to be very weak. There a numberof factors that contribute to the very weak Raman signal collected fromthe skin. One factor is the limited amount of high-intensity energy thatone can safely deliver into tissue without causing photo-damage to thetissue. A second factor is the limited Raman scattering efficiencyinherent to most molecules of analytical and physiological interest. Athird factor is the scattering and absorbance characteristics of thetissue that limit the amount of energy that can be effectively deliveredinto the tissue and the amount of Raman spectral information that can becollected from the tissue.

Another type of signal is a fluorescence signal, which like Ramansignals also has disadvantages. Fluorescence signals are more general innature than Raman signals. Fluorescence molecules of interest may be ofa smaller number than desired. The scattering and absorbancecharacteristics of the tissue limit the amount of energy that can beeffectively delivered into the tissue and the amount of fluorescencespectral information that can be collected from the tissue.

Optical absorbance and tissue scattering, which are two fundamentaloptical properties of tissue, can be transient during non-invasivedetection of an analyte such as glucose. Optical absorbance and tissuescattering can affect the glucose concentration measurement.

It would be desirable to develop a non-invasive method using spectralinformation such as Raman or fluorescence spectral information that moreaccurately determines the analyte concentration.

SUMMARY OF THE INVENTION

According to one non-invasive method, the concentration of an analyte isdetermined using Raman spectral information. A high-intensity, narrowband of light is applied to a first side of skin tissue. Thehigh-intensity light enters the skin tissue and generates a Ramansignal. A Raman-generating material is placed in a location nearest asecond side of the skin tissue. The second side is located generallyopposite of the first side. The high-intensity light is reflected fromthe Raman-generating material so as to produce additional Raman signalthat passes through the skin tissue towards the first side of the skintissue. The Raman signal generated from the high-intensity lightentering the skin tissue is reflected towards the first side of the skintissue via the Raman-generating material. The Raman signal generatedfrom the high-intensity light entering the skin tissue and theadditional Raman signal generated from the Raman-generating material iscollected. The analyte concentration using information from thecollected Raman signals is determined.

According to another non-invasive method, a diagnosis using Ramanspectral information is determined. A high-intensity, narrow band oflight is applied to a first side of skin tissue. The high-intensitylight enters the skin tissue and generates a Raman signal. ARaman-generating material is placed in a location nearest a second sideof the skin tissue. The second side is located generally opposite of thefirst side. The high-intensity light is reflected from theRaman-generating material so as to produce additional Raman signal thatpasses through the skin tissue towards the first side of the skintissue. The Raman signal generated from the high-intensity lightentering the skin tissue is reflected towards the first side of the skintissue via the Raman-generating material. The Raman signal generatedfrom the high-intensity light entering the skin tissue and theadditional Raman signal generated from the Raman-generating material iscollected. Information from the collected Raman signals is used toperform a general diagnosis.

According to a further non-invasive method, the concentration of ananalyte using Raman spectral information is determined. An area of theskin tissue is pinched. A Raman-generating material is placed near oraround the pinched skin tissue. The Raman-generating material forms atleast one opening therethrough. A high-intensity, narrow band of lightis applied to the skin tissue through the at least one opening. Thehigh-intensity light enters the skin tissue and generates a Ramansignal. The high-intensity light and Raman signal that pass through thepinched skin tissue is reflected back into the pinched skin tissue viathe Raman-generating material. The Raman signal generated from thehigh-intensity light entering the skin tissue and the additional Ramansignal generated from the Raman-generating material is collected. Theanalyte concentration is determined using the collected Raman signals.

According to a non-invasive method, the concentration of an analyteusing fluorescence spectral information is determined. A high-intensity,narrow band of light is applied to a first side of skin tissue. Thehigh-intensity light enters the skin tissue and generates a fluorescencesignal. A fluorescence-generating material is placed in a locationnearest a second side of the skin tissue. The second side is locatedgenerally opposite of the first side. The high-intensity light isreflected from the fluorescence-generating material so as to produceadditional fluorescence signal that passes through the skin tissuetowards the first side of the skin tissue. The fluorescence signalgenerated from the high-intensity light entering the skin tissue isreflected towards the first side of the skin tissue via thefluorescence-generating material. The fluorescence signal generated fromthe high-intensity light entering the skin tissue and the additionalfluorescence signal generated from the fluorescence-generating materialis collected. The analyte concentration using information from thecollected fluorescence signals is determined.

According to another non-invasive method, a diagnosis using fluorescencespectral information is performed. A high-intensity light is applied toa first side of skin tissue. The high-intensity light enters the skintissue and generates a fluorescence signal. A fluorescence-generatingmaterial is placed in a location nearest a second side of the skintissue. The second side is located generally opposite of the first side.The high-intensity light is reflected from the fluorescence-generatingmaterial so as to produce additional fluorescence signal that passesthrough the skin tissue towards the first side of the skin tissue. Thefluorescence signal generated from the high-intensity light entering theskin tissue is reflected towards the first side of the skin tissue viathe fluorescence-generating material. The fluorescence signal generatedfrom the high-intensity light entering the skin tissue and theadditional fluorescence signal generated from thefluorescence-generating material is collected. Information from thecollected fluorescence signals is used to perform a general diagnosis.

According to a further non-invasive method, the concentration of ananalyte using fluorescence spectral information is determined. An areaof the skin tissue is pinched. A fluorescence-generating material isplaced near or around the pinched skin tissue. Thefluorescence-generating material forms at least one openingtherethrough. A high-intensity, narrow band of light is applied to theskin tissue through the at least one opening. The high-intensity lightenters the skin tissue and generates a fluorescence signal. Thehigh-intensity light and fluorescence signal that pass through thepinched skin tissue back is reflected into the pinched skin tissue viathe fluorescence-generating material. The fluorescence signal generatedfrom the high-intensity light entering the skin tissue and theadditional fluorescence signal generated from thefluorescence-generating material is collected. The analyte concentrationusing the collected fluorescence signals is determined.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration used in determining the concentration of ananalyte using Raman spectral information according to one embodiment.

FIG. 2 a is an illustration further detailing the spatial filter used indetermining the analyte concentration of FIG. 1.

FIG. 2 b, 2 c depicts positions of a movable spatial filter according toone embodiment.

FIG. 3 is an illustration used in determining the analyte concentrationusing Raman spectral information according to another embodiment.

FIG. 4 is an illustration used in determining the analyte concentrationusing Raman spectral information according to another embodiment.

FIG. 5 a is an illustration used in determining the analyteconcentration using Raman spectral information according to a furtherembodiment.

FIG. 5 b is an illustration used in determining the analyteconcentration using Raman spectral information according to a furtherembodiment.

FIG. 6 a is an illustration used in determining the concentration of ananalyte using fluorescence spectral information according to anotherembodiment.

FIG. 6 b is an illustration further detailing the spatial filter used indetermining the analyte concentration of FIG. 6 a.

FIG. 7 is an illustration used in determining the analyte concentrationusing fluorescence spectral information according to a furtherembodiment.

FIG. 8 is an illustration used in determining the analyte concentrationusing fluorescence spectral information according to yet anotherembodiment.

FIG. 9 a is an illustration used in determining the analyteconcentration using fluorescence spectral information according to afurther embodiment.

FIG. 9 b is an illustration used in determining the analyteconcentration using fluorescence spectral information according to afurther embodiment.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

In one method, the invention is directed to non-invasive methods fordetermining the concentration of an analyte uses Raman spectralinformation. The invention is adapted to increase optical throughput inthese methods using spectral information. Analytes that may be measuredusing the Raman spectral information include glucose, lipid profiles(e.g., cholesterol, triglycerides, LDL and HDL), microalbumin,hemoglobin A_(1C), fructose, lactate, or bilirubin. The presentinvention is not limited, however, to these specific analytes and it iscontemplated that other analyte concentrations may be determined. Theanalytes may be in, for example, a whole blood sample, a blood serumsample, a blood plasma sample, and other body fluids like ISF(interstitial fluid) and urine.

The present invention assists in providing a method of correcting foroptical absorbance and/or tissue scattering that can be transient duringnon-invasive analyte (e.g., glucose) detection. The Raman signature ofanalytes such as glucose can be corrected based on the opticalabsorbance and tissue scattering occurring in the tissue. The absorptionof the skin tissue and tissue scattering may vary in the short term, aswell as the long term. For example, one non-limiting short-termsituation could be increased blood flow or changes in the tissuehydration. One non-limiting long-term condition could be an individual'sskin being tan or even burned. It is contemplated that the absorption ofthe skin tissue and tissue scattering may vary under other short-termand long-term conditions such as localized hematocrit, tissue deformity(e.g., scar or melanoma), temperature, pH or skin morphology.

According to one method, the concentration of an analyte is determinedusing Raman spectral information. A high-intensity, narrow band of lightis applied to a first side of skin tissue. The high-intensity lightenters the skin tissue and generates a Raman signal. A Raman-generatingmaterial is placed in a location nearest a second side of skin tissue.The second side is located generally opposite of the first side. Thehigh-intensity light from the Raman-generating material is reflected soas to produce additional Raman signal that passes through the skintissue towards the first side of the skin tissue. The Raman signalgenerated from the high-intensity light entering the skin tissue isreflected towards the first side of the skin tissue via theRaman-generating material. The Raman signal generated from thehigh-intensity light entering the skin tissue and the additional Ramansignal generated from the Raman-generating material are collected. Theanalyte concentration is determined using information from the collectedRaman signals.

Referring to FIG. 1, an illustration is used showing the location of theRaman-generating material and the high-intensity, narrow band of lightaccording to one method. High-intensity light 10 is applied to skintissue 12 such as pinched skin tissue or a finger. The high-intensitylight 10 is shown in FIG. 1 as coming from a high-intensity light source10 a. The high-intensity light source may be a variety of light sources.For example, the high-intensity light source may come from amonochromatic light source that is delivered in a narrow band. Oneexample of a monochromatic light source is a laser-diode source. It iscontemplated that other light sources may be used such as alight-emitting diode and incoherent lamps. The light sources may befiltered to provide a more clearly defined (i.e., narrow) band of light.It is also contemplated that the high-intensity light may be a dyelaser, gas laser, ion laser or a pumped laser.

The wavelength of the light source may vary but is generally from about300 to about 10,000 nm. The light source may be an ultraviolet lightsource, a near-infrared light source, an infrared light source, orvisible light source with appropriate filtering. The light source to beused would be a high-intensity, narrow band of light.

The Raman spectral information in one method may be collected in thewavelength range from about 300 nm to about 12,000 nm. However, severalwavelength-dependent characteristics unique to tissue optics and to theRaman effect can significantly impact the ability to successfully employthe Raman technique for the non-invasive determination of analytes intissue. For example, at lower wavelengths, the inherent Raman signalfrom analytes in tissue is relatively strong, but tissueautofluorescence is also relatively strong, which may overwhelm andcomplicate detecting the Raman signal in the tissue. Conversely, athigher wavelengths, tissue autofluorescence and the inherent Ramansignal decrease. The choice of the light source would be made based on abalance of the Raman signal power and the autofluorescence interferenceat the wavelengths of interest for the analyte of interest. Therefore,for glucose analysis, it is desirable to employ a high-intensity, narrowband light source centered at or near 830 nm and collect the Ramanspectral information in the wavelength range of from above 830 nm toabout 1030 nm where the strength of the Raman signal is optimized versesthe tissue autofluorescence.

The glucose-related Raman spectral information may be collected fromRaman scattered light shifted from 100 cm⁻¹ to 10,000 cm⁻¹ away from thelight source. More specifically, the glucose-related Raman spectralinformation may be collected from Raman scattered light shifted from 100cm⁻¹ to 1600 cm⁻¹ away from the light source since the strongest glucosepeaks occur at Raman shifts of about 1340 cm⁻¹ and about 1125 cm⁻¹. Itis contemplated that the Raman spectral information may be collected indifferent ranges, especially if the analyte concentration to bedetermined is not glucose.

One specific example is an 830 nm laser-diode source. One example of acommercially available 830 nm laser-diode source is Invictus™ NIR 830 nmdiode laser, which is marketed by Kaiser Optical Systems, Inc. of AnnArbor, Mich. Another example is a PI-ECL-830-300 diode laser, which ismarketed by Process Instruments of Salt Lake City, Utah. In oneembodiment, the laser light is delivered to the skin tissue in about a 1mm beam diameter. It is contemplated that other laser-diode sources maybe employed.

The high-intensity, narrow band of light may be adjusted so that ahigher resolution Raman spectrum is generated. For example, thehigh-intensity narrow band of light may be limited, resulting in lesslight being exposed and a higher resolution Raman signal being obtained.By adjusting the high-intensity, narrow band of light, the strength ofthe Raman signal and the exposure may be optimized depending on theanalyte of interest.

The high-intensity light 10 enters on a first side 12 a of the skintissue 12. The thickness of the skin tissue that may be used indetermining the analyte concentration may vary. The skin tissue isgenerally from about 1 to about 5 mm in thickness. More specifically,the skin is generally from about 1 to about 3 mm in thickness. The skintissue may be pinched when the high-intensity light enters the skintissue.

As shown in FIG. 1, the high-intensity light 10 enters the skin tissue12 at point A. After the high-intensity light 10 enters the skin tissue12, a Raman signal is generated and scatters in all directions. Aportion of the high-intensity light may contact the skin withoutentering the skin and scatter in all directions. A portion 16 of theRaman signal is redirected back towards collection optics 18 afterentering the skin tissue 12. Some of the Raman signal exits the skintissue 12, however, and is reflected back using a Raman-generatingmaterial 22. The Raman-generating material 22 reflects back Ramansignals towards the collection optics 18 that would otherwise have beenlost exiting the other side 12 b of the skin tissue 12, which isopposite of the collection optics 18. Thus, an increased fraction of theRaman signal will be redirected to the collection optics 18. It iscontemplated that an increased fraction of the Raman signal may beredirected using a reflective surface such as a mirror. A portion of theRaman signal created by the Raman-generating material is scattered atoblique angles and will not be detected or may also be absorbed beforebeing detected.

The Raman-generating material 22 is placed in a location nearest theother side 12 b of the skin tissue 12. The Raman-generating material islocated generally opposite of the entry of the applied high-intensitylight. As shown in FIG. 1, the Raman-generating material 22 is locatedopposite of the entry of the high-intensity light 10 at point A inFIG. 1. It is contemplated that the Raman-generating material may be asingle reflector as shown in FIG. 1 or a plurality of reflectors.

The Raman-generating material 22 also receives the high-intensity light10 and generates additional Raman signal therefrom. To the extent thatthe Raman-generating material does not create Raman signals from thehigh-intensity light 10, the Raman-generating material 22 reflects backthe remaining portion of the high-intensity light back through the skintissue 12. These Raman signals will typically envelop a larger volume ofskin tissue because the Raman signals will originate and scatteroutwardly from every point in the skin tissue. After this high-intensitylight is reflected back into the skin tissue 12 via the Raman-generatingmaterial 22, additional Raman signals may be generated. Thus, theoptical pathlength is increased by passing the source light through theskin tissue twice. By increasing the optical pathlength, the resultinganalytical signal is also increased.

Using the Raman-generating material 22, a measure of optical absorptioncan be obtained. Absorption is generally proportional to the totalquantity of Raman signal from the Raman-generating material 22 thatpasses through the sample. In the case where the entire Raman signal canbe integrated, then the analytical signal can be further corrected forchanges in tissue absorbance and tissue scattering. Changes in tissueabsorbance and scattering may be caused by, for example, increased bloodflow or changes in tissue hydration. For example, the intensity of theRaman signals from the Raman-generating material using only thehigh-intensity light can be compared to the intensity of the Ramansignals using the Raman-generating material and the body tissue usingthe high-intensity light. A comparison of these intensities candetermine and quantify the level of optical absorbance by the tissue.The level of optical absorbance can be done on an absolute basis or arelative basis. If done on an absolute basis, the high-intensity lightwill typically need to be adjusted to have a similar intensity levelwhen contacting the Raman-generating material 22.

Thus, in summary, the Raman-generating material 22 (a) reflects backRaman signal created on the initial pass through the skin tissue thatotherwise would have been lost; (b) creates Raman signal from thehigh-intensity light 10; and (c) reflects back the light source that didnot create a Raman signal back into the skin tissue with the possibilityof forming an additional Raman signal. These Raman signals aredesignated generally in FIG. 1 as Raman signals 20.

The Raman-generating material may be formed from a variety of materials.For example, the Raman-generating material may include a polymericmaterial such as Spectralon™ polymeric lining. Spectralon™ polymericlining is a thermoplastic resin with a very high diffuse reflectance.Spectralon™ polymeric lining is available through Labsphere Inc. ofNorth Sutton, N.H.

It is contemplated that other materials may be used as theRaman-generating material including other polymeric materials. Forexample, the Raman-generating material may include a polystyrene surfaceor polycarbonate surface. The Raman-generating material to be used needsto generate a unique Raman signal.

In one embodiment, the Raman-generating material may be a thin coatingor layer on a thicker substrate, which is not a Raman-generatingmaterial. In another embodiment, the substrate may be formed entirely ofthe Raman-generating material.

Determining the concentration of an analyte (e.g., glucose) requires ameasure proportional to the quantity of the analyte and a measure of thevolume in which that quantity analyte resides. Employing a measure ofoptical scattering allows the analyte concentration calculation to becorrected should the optical probe volume change over the course ofseveral measurements.

In one method, before collecting the Raman signals, a spatial filter isprovided that measures the optical scattering of the Raman signals inthe tissue. The amount of scattering affects the probe volume of thebody tissue. The use of a spatial filter differentiates scattering fromabsorbance and further approximates the relative changes in absorbanceand scattering in a skin tissue sample of fixed thickness.

In one method, a spatial filter is placed to block a fraction of theRaman signals emerging from the skin tissue and to allow the remainingfraction of the Raman signals to strike the at least one detector. Thus,a spatial filter blocks light in one portion and allows light through inanother portion. FIG. 1 depicts a spatial filter 80, which is shown inmore detail in FIG. 2 a. The spatial filter 80 includes a plurality ofapertures 82, 84, 86 being formed. Sections 80 a, 80 b of the spatialfilter 80 block a fraction of the Raman signals. If the opticalscattering is low, the intensity (I) of the Raman signal at the middleaperture 84 would likely be significantly higher than the intensities ofthe Raman signals at outer apertures 82, 86 since a small fraction ofthe Raman signals would take a lateral trajectory through the skintissue.

In the scenario with optical scattering being low, a ratio of theintensity of Raman light through the right aperture 86 (I_(r)) to theintensity of the Raman light through the middle aperture 84 (I_(m))after correction for path length would be small. Similarly, in thescenario with optical scattering being low, a ratio of the intensity ofRaman light through the left aperture 82 (I_(l)) to the intensity of theRaman light through the middle aperture 84 (I_(m)) after correction forpath length would also be small. Thus, when I_(r)/I_(m) and I_(l)/I_(m)are small, the optical scattering is low. The greater the scattering inthe tissue, the greater the ratios of I_(r)/I_(m) and I_(l)/I_(m). It isalso noted that the absorption of FIG. 2 a would be generallyapproximate to the sum of the intensities (I_(r)+I_(m)+I_(l)).

Using the apparatus of FIG. 2 a, if the condition of the body tissuechanges so as to affect the scattering properties of the tissue, thenthe spatial pattern of the Raman signals from the Raman-generatingmaterial would also subsequently change. By applying this relativechange to a series of analyte measurements, the analyte signal isnormalized against optical probe volume and, thus, increases theaccuracy of the calculated analyte concentration.

It is contemplated that the spatial filter may be adjustable to adjustthe size of the aperture openings. This may be advantageous todiscriminate scattered light from absorbed light. For example, referringto FIGS. 2 b and 2 c, a spatial filter 90 includes sections 90 a and 90b. The sections 90 a, 90 b are movable along a generally horizontaldirection in either direction (see arrow A). FIG. 2 b depicts thesections 90 a, 90 b in a more open position such that the distancetherebetween is represented by distance D1. FIG. 2 c depicts thesections 90 a, 90 b in a more closed position such that the distancetherebetween is represented by distance D2.

It is also contemplated that the spatial filter itself may be movable soas to better determine and characterize the scattered light. Forexample, the spatial filter may be moved to determine where the lightpropagates through the sample and/or to selectively measure multiplescattered or transmitted non-scattered light. If you excite a firstpoint and measure at a second point with an aperture, then you have someidea of pathlength. By having a better idea of the pathlength, thetissue volume can be adjusted, if necessary, with respect to the analytesignal, which results in a more accurate analyte concentration.

Referring back to FIG. 1, after the Raman signals proceed through thespatial filter 80, the collection optics 18 collect the returned Ramansignals 16, 20. It is contemplated that the collection optics maycollect the Raman signals before they pass through the spatial filter.The spatial filter 80 in this method, however, needs to be locatedbefore the Raman signals are passed to a detector.

The collected Raman signals are then passed to a detector 30. Thedetector 30 assists in determining the analyte concentration (e.g.,glucose) from the collected Raman signals. One example of a detectorthat may be used is a silicon detector. Other examples of detectorsinclude an extended InGaAs detector, a germanium detector, a leadselenide (PbSe) detector, or a lead sulfide (PbS) detector. It iscontemplated that other detectors may be employed to assist indetermining the analyte concentration (e.g., glucose) from the collectedRaman signals.

It is contemplated that a plurality of detectors and a plurality ofapertures may be used. In this embodiment, the plurality of aperturesand detectors may approximate a direct-imaging arrangement, which likelywould provide a more accurate measure of scattering and absorption.

The corrections for Raman absorption and/or scattering properties of thebody tissue related to quantifying analytes that have weak Raman signals(e.g., glucose in body tissue) may be accomplished by several methods.In one method, a calibration algorithm that incorporates absorptionand/or scattering properties of the tissue to correct for the analyteconcentration reading.

In another method, the collection times are automatically adjusted sothat the appropriate signal-to-noise ratio is achieved, which assists inobtaining a more accurate analyte reading. For example, the collectiontimes may be increased to increase the total amount of signals, whichgenerally translates to better signals, especially with smaller signals.

By correcting or accounting for the optical absorbance and/or scatteringin the skin tissue, the Raman signature of glucose can be quantitativelydetermined in a more accurate manner. Thus, this method provides anoptical solution to correct quantitative, analytical signals for changesin the tissue optical properties.

The collection optics may vary from that depicted in FIG. 1. FIG. 3depicts an illustration similar to FIG. 1 that includes a parabolicmirror 40 in which the high-intensity light 10 passes through an opening42 formed therein. The high-intensity light 10 enters the tissue andgenerates Raman signals, which scatter in all directions. The scatteredRaman signals 46 are directed back to the parabolic mirror 40 afterpassing between or around the spatial filter sections 80 a, 80 b. TheRaman signals are further reflected by the parabolic mirror to adetector 50 where the analyte concentration is determined from thecollected Raman signals. The analyte concentration in this method may becorrected in a similar manner as discussed in connection with FIGS. 1and 2.

According to another embodiment, the collection optics may be othermirrors with curvatures that deliver focused laser light back into thetissue. Alternatively, the collection optics may be other mirrors withcurvatures that are shaped to deliver parallel light back into thetissue depending on the Raman signal collection optics.

In another embodiment, the spatial distribution of the Raman signals mayalso be achieved by using an optical design based on spatial imaging. Inthis embodiment, a spatial filter is unnecessary. One such non-limitingexample is shown in FIG. 4. FIG. 4 shows the high-intensity light source10 a, the skin issue 12, and the Raman-generating material 22. Ramansignals 100 are collected by imaging optics 110 and then are directed toan array detector 112. The array detector 112 is an array of individualsdetectors (pixels) that each measure a portion of Raman signal. Theimaging optics and the detector, in one method, use the Raman signals tocorrect for both absorption and scattering caused by the tissue.

According to another method, a non-invasive method of determining theconcentration of an analyte using Raman spectral information includespinching an area of the skin tissue. An area of the skin tissue ispinched. A Raman-generating material is placed near or around thepinched skin tissue. The Raman-generating material forms at least oneopening therethrough. A high-intensity, narrow band of light is appliedto the skin tissue through the at least one opening. The high-intensitylight enters the skin tissue and generates a Raman signal. Thehigh-intensity light and Raman signal that pass through the pinched skintissue are reflected back into the pinched skin tissue via theRaman-generating material. The Raman signal generated from thehigh-intensity light entering the skin tissue and the additional Ramansignal generated from the Raman-generating material is collected. Theanalyte concentration is determined using the collected Raman signals.

As shown in FIG. 5 a, a Raman-generating material 170 is placed near oraround a pinched skin tissue 180. The width of the pinched skin tissueis generally from about 1 to about 2 mm. The Raman-generating material170 forms at least one opening 172 in which the high-intensity light 174is applied through the at least one opening 172. The high-intensitylight 174 enters the pinched skin tissue 180 and generates a Ramansignal. The high-intensity light and Raman signal that pass through thepinched skin tissue are reflected back into the pinched skin tissue viathe Raman-generating material 170. Additionally, the Raman-generatingmaterial 170 generates an additional Raman signal. The Raman signals arecollected and the analyte concentration is determined using thecollected Raman signals.

The Raman signals may be collected via high NA (numerical aperture)optics or NA fiber(s) 190. The high NA (numerical aperture) optics or NAfiber(s) 190 transmit the collected Raman signals to a spectrometer 192.It is contemplated that the collected signals may be transmitted to asingle detector with a filter, a CCD (cathode-coupled detector), a diodearray, or other devices that detect a specific signal. It iscontemplated that the Raman signals may be collected on the same side asthe high-intensity light entering the pinched skin tissue such as shown,for example, in FIG. 5 b.

In addition to determining analyte concentration, the Raman spectralinformation may be used in other methods. For example, in one method,information from the collected Raman signals may be used to perform ageneral diagnosis. The general diagnosis may include identifying (a) thepresence of a particular analyte; (b) a particular molecule or (c)tissue morphology. The general diagnosis can be directed to severalbeneficial applications. For example, potential cancerous skin lesionsmay be identified in one application. By identifying potential cancerouscells, the tissue removal can be minimized. In another application, thestage of cancerous cells may be identified. In a further application,the effectiveness of cancer photodynamic therapy may be tracked. It iscontemplated that other diagnosis may be performed using the inventivemethods.

In addition to using Raman signals, other signals may be used in othermethods. In another method, a non-invasive method for determining theconcentration of an analyte uses fluorescence spectral information.Analytes that may be measured using fluorescence spectral informationinclude glucose, lipid profiles (e.g., cholesterol, triglycerides, LDLand HDL), microalbumin, hemoglobin A_(1C), or bilirubin. The presentinvention is not limited, however, to these specific analytes and it iscontemplated that other analyte concentrations may be determined. Theanalytes may be in, for example, a whole blood sample, a blood serumsample, a blood plasma sample, and other body fluids like ISF(interstitial fluid) and urine.

The present invention assists in providing a method of correcting foroptical absorbance and/or tissue scattering that can be transient duringnon-invasive analyte (e.g., glucose) detection. The fluorescencesignature of analytes such as glucose can be corrected based on theoptical absorbance and tissue scattering occurring in the tissue. Theabsorption of the skin tissue and tissue scattering may vary in theshort term, as well as the long term as discussed above. According toanother method, the analyte concentration is determined usingfluorescence spectral information. A high-intensity band of light isapplied to a first side of skin tissue. The high-intensity light entersthe skin tissue and generates a fluorescence signal. Afluorescence-generating material is placed in a location nearest asecond side of skin tissue. The second side is located generallyopposite of the first side. The high-intensity light from thefluorescence-generating material is reflected so as to produceadditional fluorescence signal that passes through the skin tissuetowards the first side of the skin tissue. The fluorescence signalgenerated from the high-intensity light entering the skin tissue isreflected towards the first side of the skin tissue via thefluorescence-generating material. The fluorescence signal generated fromthe high-intensity light entering the skin tissue and the additionalfluorescence signal generated from the fluorescence-generating materialare collected. The analyte concentration is determined using informationfrom the collected fluorescence signals.

Referring to FIG. 6 a, an illustration is used showing the location ofthe fluorescence-generating material and the high-intensity band oflight according to one method. High-intensity light 210 is applied tothe skin tissue 12 such as pinched skin tissue or a finger. Thehigh-intensity light 210 is shown in FIG. 6 a as coming from ahigh-intensity light source 210 a. The high-intensity light 210 may be anarrow band of light, but does not necessarily have to be a narrow bandof light. The high-intensity light source may come from a monochromaticlight source. It is contemplated that other light sources may be usedsuch as a light-emitting diode, incoherent lamps, a dye laser, gaslaser, ion laser or a pumped laser.

The wavelength of the light source may vary but is generally between 300and 10,000 nm. The fluorescence spectral information in one method maybe collected in the wavelength range from about 300 nm to about 12,000nm. It is contemplated that the fluorescence spectral information may becollected in different ranges depending on the analyte concentration tobe determined.

The high-intensity light 210 enters on the first side 12 a of the skintissue 12. As shown in FIG. 6 a, the high-intensity light 210 enters theskin tissue 12 at point A. After the high-intensity light 210 enters theskin tissue 12, a fluorescence signal is generated and scatters in alldirections. A portion of the high-intensity light may contact the skinwithout entering the skin and scatter in all directions. A portion 216of the fluorescence signal is redirected back towards the collectionoptics 18 after entering the skin tissue 12. Some of the fluorescencesignal exits the skin tissue 12, however, and is reflected back using afluorescence-generating material 222. The fluorescence-generatingmaterial 222 reflects back fluorescence signals towards the collectionoptics 18 that would otherwise have been lost exiting the other side 12b of the skin tissue 12, which is opposite of the collection optics 18.Thus, an increased fraction of the fluorescence signal will beredirected to the collection optics 18. It is contemplated that anincreased fraction of the fluorescence signal may be redirected using areflective surface such as a mirror. A portion of the fluorescencesignal created by the fluorescence-generating material is scattered atoblique angles and will not be detected or may also be absorbed beforebeing detected.

The fluorescence-generating material 222 is placed in a location nearestthe other side 12 b of the skin tissue 12. The fluorescence-generatingmaterial is located generally opposite of the entry of the appliedhigh-intensity light. As shown in FIG. 6 a, the fluorescence-generatingmaterial 222 is located opposite of the entry of the high-intensitylight 210 at point A in FIG. 6 a. It is contemplated that thefluorescence-generating material may be a single reflector as shown inFIG. 6 a or a plurality of reflectors.

The fluorescence-generating material 222 also receives thehigh-intensity light 210 and generates additional fluorescence signaltherefrom. To the extent that the fluorescence-generating material doesnot create fluorescence signals from the high-intensity light 210, thefluorescence-generating material 222 reflects back the remaining portionof the high-intensity light back through the skin tissue 12. Thesefluorescence signals will typically envelop a larger volume of skintissue because the fluorescence signals will originate and scatteroutwardly from every point in the skin tissue. After this high-intensitylight is reflected back into the skin tissue 12 via thefluorescence-generating material 222, additional fluorescence signalsmay be generated. Thus, the optical pathlength is increased by passingthe source light through the skin tissue twice. By increasing theoptical pathlength, the resulting analytical signal is also increased.

Using the fluorescence-generating material 222, a measure of opticalabsorption can be obtained. Absorption is generally proportional to thetotal quantity of fluorescence signal from the fluorescence-generatingmaterial 222 that passes through the sample. In the case where theentire fluorescence signal can be integrated, then the analytical signalcan be further corrected for changes in tissue absorbance and tissuescattering. In the case where the entire Raman signal can be integrated,then the analytical signal can be further corrected for changes intissue absorbance and scattering. Changes in tissue absorbance andscattering may be caused by, for example, increased blood flow orchanges in tissue hydration. For example, the intensity of thefluorescence signals from the fluorescence-generating material usingonly the high-intensity light can be compared to the intensity of thefluorescence signals using the fluorescence-generating material and thebody tissue using the high-intensity light. A comparison of theseintensities can determine and quantify the level of optical absorbanceby the tissue.

Thus, in summary, the fluorescence-generating material 222 (a) reflectsback fluorescence signal created on the initial pass through the skintissue that otherwise would have been lost; (b) creates fluorescencesignal from the high-intensity light 210; and (c) reflects back thelight source that did not create a fluorescence signal back into theskin tissue with the possibility of forming an additional fluorescencesignal. These fluorescence signals are designated generally in FIG. 6 aas fluorescence signals 220.

The fluorescence-generating material may function under a fluorescencemechanism where light of a shorter wavelength excites the molecule andthen the molecule fluoresces, giving off light of a longer wavelength.The fluorescence-generating material may be formed from a variety ofmaterials such fluorescence dyes. The fluorescence dyes may benear-infrared (NIR) dyes, IR dyes and visible dyes. Some examples of NIRfluorescent dyes include derivatives from cyanine dyes (Cy5.5) or theclinically-approved indocyanine green (ICG). Such dyes are typicallyused as a coating since they are usually aqueous in nature. The dyes maybe mobilized or impregnated in the fluorescence-generating material. Itis contemplated that other materials may be used as afluorescence-generating material that function in a similar manner.

Other materials that fluoresce in the NIR operate slightly differently.The material used in the NIR card functions on “photon upconversion”where light of a longer wavelength is absorbed by a first molecule andthe energy is transferred to a second molecule that fluoresces at ashorter wavelength. This process is referred to upconversion since theexcitation light is of lower energy than the emission light. One exampleis the use of quantum dots. Quantum dots are small metallic materialswhose fluorescence is size dependent. It is contemplated that othermaterials may be used as a fluorescence-generating material thatfunction in a similar manner.

It is contemplated that other materials may be used as thefluorescence-generating material that fluoresce in a different mannerand in a different spectrum. The fluorescence-generating material to beused generates a unique fluorescence signal.

In one embodiment, the fluorescence-generating material may be a thincoating or layer on a thicker substrate, which is not afluorescence-generating material. In another embodiment, the substratemay be formed entirely of the fluorescence-generating material.

Determining the concentration of an analyte (e.g., glucose) requires ameasure proportional to the quantity of the analyte and a measure of thevolume in which that quantity analyte resides. Employing a measure ofoptical scattering allows the analyte concentration calculation to becorrected should the optical probe volume change over the course ofseveral measurements.

In one method, before collecting the fluorescence signals, a spatialfilter is provided that measures the optical scattering of thefluorescence signals in the tissue. In one method, a spatial filter isplaced to block a fraction of the fluorescence signals emerging from theskin tissue and to allow the remaining fraction of the fluorescencesignals to strike the at least one detector. Thus, a spatial filterblocks light in one portion and allows light through in another portion.FIG. 6 a depicts a spatial filter 280, which is shown in more detail inFIG. 6 b. The spatial filter 280 includes a plurality of apertures 282,284, 286 being formed. Sections 280 a, 280 b of the spatial filter 280block a fraction of the fluorescence signals. If the optical scatteringis low, the intensity (I) of the fluorescence signal at the middleaperture 284 would likely be significantly higher than the intensitiesof the fluorescence signals at outer apertures 282, 286 since a smallfraction of the fluorescence signals would take a lateral trajectorythrough the skin tissue.

In the scenario with optical scattering being low, a ratio of theintensity of fluorescence light through the right aperture 286 (I_(r))to the intensity of the fluorescence light through the middle aperture284 (I_(m)) after correction for path length would be small. Similarly,in the scenario with optical scattering being low, a ratio of theintensity of fluorescence light through the left aperture 282 (I_(l)) tothe intensity of the fluorescence light through the middle aperture 284(I_(m)) after correction for path length would also be small. Thus, whenI_(r)/I_(m) and I_(l)/I_(m) are small, the scattering is low. Thegreater the scattering in the tissue, the greater the ratios ofI_(r)/I_(m) and I_(l)/I_(m). It is also noted that the absorption ofFIG. 6 b would be generally approximate to the sum of the intensities(I_(r)+I_(m)+I_(l)).

Using the apparatus of FIG. 6 b, if the condition of the body tissuechanges so as to affect the scattering properties of the tissue, thenthe spatial pattern of the fluorescence signals from thefluorescence-generating material would also subsequently change. Byapplying this relative change to a series of analyte measurements, theanalyte signal is normalized against optical probe volume and, thus,increases the accuracy of the calculated analyte concentration.

It is contemplated that the spatial filter may be adjustable to adjustthe size of the aperture openings as, for example, described above inthe spatial filter 90. It is also contemplated that the spatial filteritself may be movable so as to better determine and locate the scatter.

The returned fluorescence signals 216, 220 are collected by thecollection optics 18. The collected fluorescence signals are then passedto a detector 230. The detector 230 assists in determining the analyteconcentration (e.g., glucose) from the collected fluorescence signals.One example of a detector of fluorescence signals that may be used is asilicon detector. Other examples of detectors include an extended InGaAsdetector, a germanium detector, a lead selenide (PbSe) detector, or alead (PbS) detector. It is contemplated that other detectors may beemployed to assist in determining the analyte concentration (e.g.,glucose) from the collected fluorescence signal.

It is contemplated that a plurality of detectors and a plurality ofapertures may be used. In this embodiment, the plurality of aperturesand detectors may approximate a direct-imaging arrangement, which likelywould provide a more accurate measure of scattering and absorption.

The corrections for fluorescence absorption and/or scattering propertiesof the body tissue related to quantifying analytes that have weakfluorescence signals (e.g., glucose in body tissue) may be accomplishedby several methods. In one method, a calibration algorithm thatincorporates absorption and/or scattering properties of the tissue tocorrect for the analyte concentration reading.

In another method, the collection times are automatically adjusted sothat the appropriate signal-to-noise ratio is achieved, which assists inobtaining a more accurate analyte reading. For example, the collectiontimes may be increased to increase the total amount of signals, whichgenerally translates to better signals, especially with smaller signals.

By correcting or accounting for the optical absorbance and/or scatteringin the skin tissue, the fluorescence signature of glucose can bequantitatively determined in a more accurate manner. Thus, this methodprovides an optical solution to correct quantitative, analytical signalsfor changes in the tissue optical properties.

The collection optics may vary from that depicted in FIG. 6 a. FIG. 7depicts an illustration similar to FIG. 6 a that includes a parabolicmirror 240 in which the high-intensity light 210 passes through anopening 242 formed therein. The high-intensity light 210 enters thetissue and generates fluorescence signals, which scatter in alldirections. The scattered fluorescence signals 246 are directed back tothe parabolic mirror 240 after passing through the spatial filtersections 280 a, 280 b. The fluorescence signals are further reflected bythe parabolic mirror to the detector 250 where the analyte concentrationis determined from the collected fluorescence signals. The analyteconcentration in this method may be corrected in a similar manner asdiscussed in connection with FIGS. 6 a, 6 b.

According to another embodiment, the collection optics may be othermirrors with curvatures that deliver focused laser light back into thetissue. Alternatively, the collection optics may be other mirrors withcurvatures that are shaped to deliver parallel light back into thetissue depending on the fluorescence-signal collection optics.

In another embodiment, the spatial distribution of the fluorescencesignals may also be achieved by using an optical design based on spatialimaging. In this embodiment, a spatial filter is unnecessary. One suchnon-limiting example is shown in FIG. 8. FIG. 8 shows the high-intensitylight source 210 a, the skin issue 12, and the fluorescence-generatingmaterial 222. Fluorescence signals 300 are collected by imaging optics310 and then are directed to the array detector 312. The array detector312 is an array of individuals detectors (pixels) that each measure aportion of fluorescence signal. The imaging optics and the detector, inone method, use the fluorescence signals to correct for both absorptionand scattering caused by the tissue.

As shown in FIG. 9 a, a fluorescence-generating material 370 is placednear or around a pinched skin tissue 380. The width of the pinched skintissue is generally from about 1 to about 2 mm. Thefluorescence-generating material 370 forms at least one opening 372 inwhich the high-intensity light 374 is applied through the at least oneopening 372. The high-intensity light 374 enters the pinched skin tissue380 and generates a fluorescence signal. The high-intensity light andfluorescence signal that pass through the pinched skin tissue arereflected back into the pinched skin tissue via thefluorescence-generating material 370. Additionally, thefluorescence-generating material 370 generates an additionalfluorescence signal. The fluorescence signals are collected and theanalyte concentration is determined using the collected fluorescencesignals.

The fluorescence signals may be collected via high NA (numericalaperture) optics or NA fiber(s) 390. The high NA (numerical aperture)optics or NA fiber(s) 390 transmit the collected fluorescence signals toa spectrometer 392. It is contemplated that the collected signals may betransmitted to a single detector with a filter, a CCD (cathode-coupleddetector), a diode array, or other devices that detect a specificsignal. It is contemplated that the fluorescence signals may becollected on the same side as the high-intensity light entering thepinched skin tissue such as shown, for example, in FIG. 9 b.

In addition to determining analyte concentration, the fluorescencespectral information may be used in other methods. For example, in onemethod, information from the collected fluorescence signals may be usedto perform a general diagnosis. The general diagnosis may includeidentifying (a) the presence of a particular analyte; (b) a particularmolecule or (c) tissue morphology. The general diagnosis can be directedto several beneficial applications. For example, potential cancerousskin lesions may be identified in one application. By identifyingpotential cancerous cells, the tissue removal can be minimized. Inanother application, the stage of cancerous cells may be identified. Ina further application, the effectiveness of cancer photodynamic therapymay be tracked. It is contemplated that other diagnosis may be performedusing the inventive methods.

Process A

A non-invasive method of determining the concentration of an analyteusing Raman spectral information, the method comprising the acts of:

-   -   applying a high-intensity, narrow band of light to a first side        of skin tissue, the high-intensity light entering the skin        tissue and generating a Raman signal;    -   placing a Raman-generating material in a location nearest a        second side of the skin tissue, the second side being located        generally opposite of the first side;    -   reflecting the high-intensity light from the Raman-generating        material so as to produce additional Raman signal that passes        through the skin tissue towards the first side of the skin        tissue;    -   reflecting the Raman signal generated from the high-intensity        light entering the skin tissue towards the first side of the        skin tissue via the Raman-generating material;    -   collecting the Raman signal generated from the high-intensity        light entering the skin tissue and the additional Raman signal        generated from the Raman-generating material; and    -   determining the analyte concentration using information from the        collected Raman signals.        Process B

The method of Process A wherein the high-intensity light is applied froma monochromatic light source.

Process C

The method of Process B wherein the high-intensity light is applied froma laser-diode source.

Process D

The method of Process A wherein the high-intensity light is applied froma light-emitting diode, a zeon-arc lamp, a dye laser, a gas-lasersource, an ion-laser source, or a pumped solid-state laser source.

Process E

The method of Process A wherein the analyte is glucose.

Process F

The method of Process A wherein the skin tissue is from about 1 to about5 mm in thickness.

Process G

The method of Process A wherein the Raman-generated material is locatedopposite of the entry of the applied high-intensity light.

Process H

The method of Process A wherein the Raman-generated material includes apolymeric material.

Process I

The method of Process H wherein the polymeric material is polystyrene orpolycarbonate.

Process J

The method of Process A further including a detector that assists indetermining the analyte concentration.

Process K

The method of Process A wherein the Raman signal is collected using atleast one parabolic mirror.

Process L

The method of Process A wherein the Raman signal is collected at awavelength range of from about 300 to about 5000 nm.

Process M

The method of Process L wherein the Raman signal is collected awavelength range of from about 830 to about 1030 nm.

Process N

The method of Process A wherein the high-intensity light is infraredlight.

Process O

The method of Process A wherein the high-intensity light isnear-infrared light.

Process P

The method of Process A wherein the high-intensity light is ultravioletlight.

Process Q

The method of Process A wherein the information from the collected Ramansignals assists in determining the level of optical scattering andabsorbance of the skin tissue, the information being used to assist indetermining the analyte concentration.

Process R

The method of Process A wherein the information from the collected Ramansignals assists in determining the level of optical scattering of theskin tissue, the information being used to assist in determining theanalyte concentration.

Process S

The method of Process A wherein the information from the collected Ramansignals assists in determining the level of absorbance of the skintissue, the information being used to assist in determining the analyteconcentration.

Process T

The method of Process A further including providing a spatial filter toassist in determining the level of absorbance of the skin tissue.

Process U

The method of Process A further including an array detector and imagingoptics.

Process V

A non-invasive method of diagnosis using Raman spectral information, themethod comprising the acts of:

-   -   applying a high-intensity, narrow band of light to a first side        of skin tissue, the high-intensity light entering the skin        tissue and generating a Raman signal;    -   placing a Raman-generating material in a location nearest a        second side of the skin tissue, the second side being located        generally opposite of the first side;    -   reflecting the high-intensity light from the Raman-generating        material so as to produce additional Raman signal that passes        through the skin tissue towards the first side of the skin        tissue;    -   reflecting the Raman signal generated from the high-intensity        light entering the skin tissue towards the first side of the        skin tissue via the Raman-generating material;    -   collecting the Raman signal generated from the high-intensity        light entering the skin tissue and the additional Raman signal        generated from the Raman-generating material; and    -   using information from the collected Raman signals to perform a        general diagnosis.        Process W

The method of Process V wherein performing the general diagnosisincludes identifying the presence of a particular analyte.

Process X

The method of Process V wherein performing the general diagnosisincludes identifying a particular molecule.

Process Y

The method of Process V wherein the high-intensity light is applied froma monochromatic light source.

Process Z

The method of Process Y wherein the high-intensity light is applied froma laser-diode source.

Process AA

The method of Process V wherein the high-intensity light is applied froma light-emitting diode, a zeon-arc lamp, a dye laser, a gas-lasersource, an ion-laser source, or a pumped solid-state laser source.

Process BB

The method of Process V wherein the analyte is glucose.

Process CC

The method of Process V wherein the skin tissue is from about 1 to about5 mm in thickness.

Process DD

The method of Process V wherein the Raman-generated material is locatedopposite of the entry of the applied high-intensity light.

Process EE

The method of Process V wherein the Raman-generated material includes apolymeric material.

Process FF

The method of Process EE wherein the polymeric material is polystyreneor polycarbonate.

Process GG

The method of Process V further including a detector that assists inperforming the general diagnosis.

Process HH

The method of Process V wherein the Raman signal is collected using atleast one parabolic mirror.

Process II

The method of Process V wherein the Raman signal is collected at awavelength range of from about 300 to about 5000 nm.

Process JJ

The method of Process II wherein the Raman signal is collected awavelength range of from about 830 to about 1030 nm.

Process KK

The method of Process V wherein the high-intensity light is infraredlight.

Process LL

The method of Process V wherein the high-intensity light isnear-infrared light.

Process MM

The method of Process V wherein the high-intensity light is ultravioletlight.

Process NN

The method of Process V wherein the information from the collected Ramansignals assists in determining the level of optical scattering andabsorbance of the skin tissue, the information being used to assist inperforming the general diagnosis.

Process OO

The method of Process V wherein the information from the collected Ramansignals assists in determining the level of optical scattering of theskin tissue, the information being used to assist in performing thegeneral diagnosis.

Process PP

The method of Process V wherein the information from the collected Ramansignals assists in determining the level of absorbance of the skintissue, the information being used to assist in determining the analyteconcentration.

Process QQ

The method of Process V further including providing a spatial filter toassist in determining the level of absorbance of the skin tissue.

Process RR

The method of Process V further including an array detector and imagingoptics.

Process SS

A non-invasive method of determining the concentration of an analyteusing Raman spectral information, the method comprising the acts of:

-   -   pinching an area of the skin tissue;    -   placing a Raman-generating material near or around the pinched        skin tissue, the Raman-generating material forming at least one        opening therethrough;    -   applying a high-intensity, narrow band of light to the skin        tissue through the at least one opening, the high-intensity        light entering the skin tissue and generating a Raman signal;    -   reflecting the high-intensity light and Raman signal that pass        through the pinched skin tissue back into the pinched skin        tissue via the Raman-generating material;    -   collecting the Raman signal generated from the high-intensity        light entering the skin tissue and the additional Raman signal        generated from the Raman-generating material; and    -   determining the analyte concentration using the collected Raman        signals.        Process TT

The method of Process SS wherein the Raman signal is collected awavelength range of from about 300 to about 5000 nm.

Process UU

The method of Process TT wherein the Raman signal is collected awavelength range of from about 830 to about 1030 nm.

Process VV

The method of Process SS wherein the analyte is glucose.

Process WW

The method of Process SS wherein the Raman-generated material includes apolymeric material.

Process XX

A non-invasive method of determining the concentration of an analyteusing fluorescence spectral information, the method comprising the actsof:

-   -   applying a high-intensity, narrow band of light to a first side        of skin tissue, the high-intensity light entering the skin        tissue and generating a fluorescence signal;    -   placing a fluorescence-generating material in a location nearest        a second side of the skin tissue, the second side being located        generally opposite of the first side;    -   reflecting the high-intensity light from the        fluorescence-generating material so as to produce additional        fluorescence signal that passes through the skin tissue towards        the first side of the skin tissue;    -   reflecting the fluorescence signal generated from the        high-intensity light entering the skin tissue towards the first        side of the skin tissue via the fluorescence-generating        material;    -   collecting the fluorescence signal generated from the        high-intensity light entering the skin tissue and the additional        fluorescence signal generated from the fluorescence-generating        material; and    -   determining the analyte concentration using information from the        collected fluorescence signals.        Process YY

The method of Process XX wherein the high-intensity light is appliedfrom a monochromatic light source.

Process ZZ

The method of Process YY wherein the high-intensity light is appliedfrom a laser-diode source.

Process AAA

The method of Process XX wherein the high-intensity light is appliedfrom a light-emitting diode or a zeon-arc lamp.

Process BBB

The method of Process XX wherein the high-intensity light is appliedfrom a dye laser, a gas-laser source, an ion-laser source, or a pumpedsolid-state laser source.

Process CCC

The method of Process XX wherein the skin tissue is from about 1 toabout 5 mm in thickness.

Process DDD

The method of Process XX wherein the fluorescence-generated material islocated opposite of the entry of the applied high-intensity light.

Process EEE

The method of Process XX wherein the fluorescence-generated materialincludes a fluorescence dye.

Process FFF

The method of Process XX wherein the fluorescence-generated materialincludes quantum dots.

Process GGG

The method of Process XX further including a detector that assists indetermining the analyte concentration.

Process HHH

The method of Process XX wherein the fluorescence signal is collectedusing at least one parabolic mirror.

Process III

The method of Process XX wherein the fluorescence signal is collected ata wavelength range of from about 300 to about 5000 nm.

Process JJJ

The method of Process III wherein the fluorescence signal is collected awavelength range of from about 830 to about 1030 nm.

Process KKK

The method of Process XX wherein the high-intensity light is infraredlight.

Process LLL

The method of Process XX wherein the high-intensity light is nearinfrared light.

Process MMM

The method of Process XX wherein the high-intensity light is ultravioletlight.

Process NNN

The method of Process XX wherein the information from the collectedfluorescence signals assists in determining the level of opticalscattering and absorbance of the skin tissue, the information being usedto assist in determining the analyte concentration.

Process OOO

The method of Process XX wherein the information from the collectedfluorescence signals assists in determining the level of opticalscattering of the skin tissue, the information being used to assist indetermining the analyte concentration.

Process PPP

The method of Process XX wherein the information from the collectedfluorescence signals assists in determining the level of absorbance ofthe skin tissue, the information being used to assist in determining theanalyte concentration.

Process QQQ

The method of Process XX further including providing a spatial filter toassist in determining the level of absorbance of the skin tissue.

Process RRR

The method of Process XX further including an array detector and imagingoptics.

Process SSS

A non-invasive method of diagnosis using fluorescence spectralinformation, the method comprising the acts of:

-   -   applying a high-intensity light to a first side of skin tissue,        the high-intensity light entering the skin tissue and generating        a fluorescence signal;    -   placing a fluorescence-generating material in a location nearest        a second side of the skin tissue, the second side being located        generally opposite of the first side;    -   reflecting the high-intensity light from the        fluorescence-generating material so as to produce additional        fluorescence signal that passes through the skin tissue towards        the first side of the skin tissue;    -   reflecting the fluorescence signal generated from the        high-intensity light entering the skin tissue towards the first        side of the skin tissue via the fluorescence-generating        material;    -   collecting the fluorescence signal generated from the        high-intensity light entering the skin tissue and the additional        fluorescence signal generated from the fluorescence-generating        material; and    -   using information from the collected fluorescence signals to        perform a general diagnosis.        Process TTT

The method of Process SSS wherein performing the general diagnosisincludes identifying the presence of a particular analyte.

Process UUU

The method of Process SSS wherein performing the general diagnosisincludes identifying a particular molecule.

Process VVV

The method of Process SSS wherein the high-intensity light is a narrowband of light.

Process WWW

The method of Process SSS wherein the high-intensity light is appliedfrom a laser-diode source.

Process XXX

The method of Process SSS wherein the high-intensity light is appliedfrom a light-emitting diode or a zeon-arc lamp.

Process YYY

The method of Process SSS wherein the high-intensity light is appliedfrom a dye laser, a gas-laser source, an ion-laser source, or a pumpedsolid-state laser source.

Process ZZZ

The method of Process SSS wherein the skin tissue is from about 1 toabout 5 mm in thickness.

Process AAAA

The method of Process SSS wherein the fluorescence-generated material islocated opposite of the entry of the applied high-intensity light.

Process BBBB

The method of Process SSS wherein the fluorescence-generated materialincludes a fluorescence dye.

Process CCCC

The method of Process SSS wherein the fluorescence-generated materialincludes quantum dots.

Process DDDD

The method of Process SSS further including a detector that assists indetermining the analyte concentration.

Process EEEE

The method of Process SSS wherein the fluorescence signal is collectedusing at least one parabolic mirror.

Process FFFF

The method of Process SSS wherein the fluorescence signal is collectedat a wavelength range of from about 300 to about 5000 nm.

Process GGGG

The method of Process FFFF wherein the fluorescence signal is collecteda wavelength range of from about 830 to about 1030 nm.

Process HHHH

The method of Process SSS wherein the information from the collectedRaman signals assists in determining the level of optical scattering andabsorbance of the skin tissue, the information being used to assist indetermining the analyte concentration.

Process IIII

The method of Process SSS wherein the information from the collectedRaman signals assists in determining the level of optical scattering ofthe skin tissue, the information being used to assist in determining theanalyte concentration.

Process JJJJ

The method of Process SSS wherein the information from the collectedRaman signals assists in determining the level of absorbance of the skintissue, the information being used to assist in determining the analyteconcentration.

Process KKKK

The method of Process SSS further including providing a spatial filterto assist in determining the level of absorbance of the skin tissue.

Process LLLL

The method of Process SSS further including an array detector andimaging optics.

Process MMMM

A non-invasive method of determining the concentration of an analyteusing fluorescence spectral information, the method comprising the actsof:

-   -   pinching an area of the skin tissue;    -   placing a fluorescence-generating material near or around the        pinched skin tissue, the fluorescence-generating material        forming at least one opening therethrough;    -   applying a high-intensity, narrow band of light to the skin        tissue through the at least one opening, the high-intensity        light entering the skin tissue and generating a fluorescence        signal;    -   reflecting the high-intensity light and fluorescence signal that        pass through the pinched skin tissue back into the pinched skin        tissue via the fluorescence-generating material;    -   collecting the fluorescence signal generated from the        high-intensity light entering the skin tissue and the additional        fluorescence signal generated from the fluorescence-generating        material; and    -   determining the analyte concentration using the collected        fluorescence signals.        Process NNNN

The method of Process MMMM wherein the fluorescence signal is collecteda wavelength range of from about 300 to about 5000 nm.

Process OOOO

The method of Process NNNN wherein the fluorescence signal is collecteda wavelength range of from about 830 to about 1030 nm.

Process PPPP

The method of Process MMMM wherein the analyte is glucose.

While the present invention has been described with reference to one ormore particular embodiments, those skilled in the art will recognizethat many changes may be made thereto without departing from the spiritand scope of the present invention. Each of these embodiments, andobvious variations thereof, is contemplated as falling within the spiritand scope of the invention as defined by appended claims.

1. A non-invasive method of determining the concentration of an analyte using Raman spectral information, the method comprising the acts of: applying a high-intensity, narrow band of light to a first side of tissue, the high-intensity light entering the tissue and generating a Raman signal; placing a Raman-generating material in a location nearest a second side of the tissue, the second side being located generally opposite of the first side; reflecting the high-intensity light from the Raman-generating material so as to produce an additional Raman signal that passes through the tissue towards the first side of the tissue; reflecting the Raman signal generated from the high-intensity light entering the tissue towards the first side of the tissue via the Raman-generating material; collecting the Raman signal generated from the high-intensity light entering the tissue and the additional Raman signal generated from the Raman-generating material; and determining the analyte concentration using information from the collected Raman signals.
 2. The method of claim 1, wherein the high-intensity light is applied from a monochromatic light source.
 3. The method of claim 2, wherein the high-intensity light is applied from a laser-diode source.
 4. The method of claim 1, wherein the analyte is glucose.
 5. The method of claim 1, wherein the tissue is skin, and the skin tissue is from about 1 to about 5 mm in thickness.
 6. The method of claim 1, wherein the Raman signal is collected at a wavelength range of from about 300 to about 5000 nm.
 7. The method of claim 6, wherein the Raman signal is collected at a wavelength range of from about 830 to about 1030 nm.
 8. The method of claim 1, wherein the information from the collected Raman signals assists in determining the level of optical scattering and absorbance of the tissue, the information being used to assist in determining the analyte concentration.
 9. The method of claim 1, wherein the information from the collected Raman signals assists in determining the level of optical scattering of the tissue, the information being used to assist in determining the analyte concentration.
 10. The method of claim 1, wherein the information from the collected Raman signals assists in determining the level of absorbance of the tissue, the information being used to assist in determining the analyte concentration.
 11. A non-invasive method of diagnosis using Raman spectral information, the method comprising the acts of: applying a high-intensity, narrow band of light to a first side of tissue, the high-intensity light entering the tissue and generating a Raman signal; placing a Raman-generating material in a location nearest a second side of the tissue, the second side being located generally opposite of the first side; reflecting the high-intensity light from the Raman-generating material so as to produce an additional Raman signal that passes through the tissue towards the first side of the tissue; reflecting the Raman signal generated from the high-intensity light entering the tissue towards the first side of the tissue via the Raman-generating material; collecting the Raman signal generated from the high-intensity light entering the tissue and the additional Raman signal generated from the Raman-generating material; and using information from the collected Raman signals to perform a general diagnosis.
 12. The method of claim 11, wherein performing the general diagnosis includes identifying the presence of a particular analyte.
 13. The method of claim 11, wherein performing the general diagnosis includes identifying a particular molecule.
 14. The method of claim 11, wherein the Raman signal is collected at a wavelength range of from about 300 to about 5000 nm.
 15. The method of claim 14, wherein the Raman signal is collected a wavelength range of from about 830 to about 1030 nm.
 16. The method of claim 11, wherein the information from the collected Raman signals assists in determining the level of optical scattering and absorbance of the skin tissue, the information being used to assist in performing the general diagnosis.
 17. The method of claim 11, wherein the information from the collected Raman signals assists in determining the level of optical scattering of the tissue, the information being used to assist in performing the general diagnosis.
 18. The method of claim 11, wherein the information from the collected Raman signals assists in determining the level of absorbance of the tissue, the information being used to assist in determining the analyte concentration.
 19. A non-invasive method of determining the concentration of an analyte using Raman spectral information, the method comprising the acts of: pinching an area of skin tissue; placing a Raman-generating material near or around the pinched skin tissue, the Raman-generating material forming at least one opening therethrough; applying a high-intensity, narrow band of light to the skin tissue through the at least one opening, the high-intensity light entering the skin tissue and generating a Raman signal; reflecting the high-intensity light and Raman signal that pass through the pinched skin tissue back into the pinched skin tissue via the Raman-generating material; collecting the Raman signal generated from the high-intensity light entering the skin tissue and an additional Raman signal generated from the Raman-generating material; and determining the analyte concentration using the collected Raman signals.
 20. The method of claim 19, wherein the Raman signal is collected a wavelength range of from about 300 to about 5000 nm.
 21. The method of claim 20, wherein the Raman signal is collected a wavelength range of from about 830 to about 1030 nm.
 22. A non-invasive method of determining the concentration of an analyte using fluorescence spectral information, the method comprising the acts of: applying a high-intensity, narrow band of light to a first side of tissue, the high-intensity light entering the tissue and generating a fluorescence signal; placing a fluorescence-generating material in a location nearest a second side of the tissue, the second side being located generally opposite of the first side; reflecting the high-intensity light from the fluorescence-generating material so as to produce an additional fluorescence signal that passes through the tissue towards the first side of the tissue; reflecting the fluorescence signal generated from the high-intensity light entering the tissue towards the first side of the tissue via the fluorescence-generating material; collecting the fluorescence signal generated from the high-intensity light entering the tissue and the additional fluorescence signal generated from the fluorescence-generating material; and determining the analyte concentration using information from the collected fluorescence signals.
 23. A non-invasive method of diagnosis using fluorescence spectral information, the method comprising the acts of: applying a high-intensity light to a first side of tissue, the high-intensity light entering the tissue and generating a fluorescence signal; placing a fluorescence-generating material in a location nearest a second side of the tissue, the second side being located generally opposite of the first side; reflecting the high-intensity light from the fluorescence-generating material so as to produce an additional fluorescence signal that passes through the tissue towards the first side of the tissue; reflecting the fluorescence signal generated from the high-intensity light entering the tissue towards the first side of the tissue via the fluorescence-generating material; collecting the fluorescence signal generated from the high-intensity light entering the tissue and the additional fluorescence signal generated from the fluorescence-generating material; and using information from the collected fluorescence signals to perform a general diagnosis. 